1. Field of the Invention
The present invention relates to methods for producing heterologous polypeptides in cyclohexadepsipeptide-deficient filamentous fungal mutant cells. The present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells. The present invention also relates to isolated cyclohexadepsipeptide synthetases and isolated nucleic acid sequences encoding the cyclohexadepsipeptide synthetases. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the cyclohexadepsipeptide synthetases. The present invention further relates to cyclohexadepsipeptides produced by the cyclohexadepsipeptide synthetases.
2. Description of the Related Art
Depsipeptides constitute a large class of peptide-related compounds derived from hydroxy and amino acids joined by amide and ester linkages. Many members of this class of compounds are biologically active and include antibiotics, alkaloids, and proteins (Shemyakin et al., 1969, Journal of Membrane Biology 1: 402-430). Examples include the enniatins, beauvericin, and bassianolide.
Enniatins are cyclohexadepsipeptide phytoxins with ionophoretic properties produced by various species of actinomycetes and filamentous fungi, particularly strains of Fusarium. They are composed of alternating D-2-hydroxyisovaleric acid residues and L-amino acids or N-methyl-L-amino acids to form an 18-membered cyclic structure and may contain more than one species of amino acid.
The biosynthesis of enniatins is catalyzed by enniatin synthetase, which is a large multifunctional enzyme that has all the essential functions for assembling enniatins from their primary precursors, i.e., D-2-hydroxyisovaleric acid, a branched chain L-amino acid (e.g., valine, leucine, isoleucine), S-adenosylmethionine, and ATP (Reper et al., 1995, European Journal of Biochemistry 230: 119-126). The precursors (D-2-hydroxyisovaleric acid and branched chain L-amino acid) are activated as thioesters. Covalently bound substrate amino acid residues are methylated under the consumption of S-adenosylmethionine. Then peptide bond formation and cyclization reactions occur.
Enniatins are postulated to play a role in wilt toxic events during infection by enniatin-producing fusaria (Walton, 1990, Biochemistry of Peptide Antibiotics, H. Kleinkauf and H. von Dohren, editors, W. de Gruytre, Berlin, pp. 179-203), and also exhibit entomopathogenic properties (Grove and Pople, 1980, Mycopathologia 70: 103-105).
The enniatin synthetase gene (esynl) has been cloned from Fusarium scirpi (Haese et al., 1993, Molecular Microbiology 7: 905-914).
Enniatin synthetase mutants of Fusarium avenaceum have been generated that do not produce enniatins (Herrmann et al., 1996, Molecular Plant-Microbe Interactions 9: 226-232).
It is an object of the present invention to provide methods for producing heterologous polypeptides in cyclohexadepsipeptide-deficient filamentous fungal mutant cells.